1. Field of the Invention
This invention generally relates to methods of inhibiting activating transcription factor/cAMP-responsive element-binding protein (ATF/CREB) and cancer cell growth and to pharmaceutical compositions for use in the methods. More specifically, this invention relates to methods of use and pharmaceutical compositions for treating certain cancers with a combination of a thiuram disulfide and metal ion or disulfiram chelated with a metal ion.
2. Description of the Prior Art
Cancer, the uncontrolled growth of malignant cells, is a major health problem of the modern medical era. While some malignancies, such as adenocarcinoma of the breast and lymphomas such as Hodgkin's Disease, respond relatively well to current chemotherapeutic antineoplastic drug regimens, other cancers are poorly responsive to chemotherapy, especially melanoma, non-small cell lung, pancreatic, liver, prostate and colon cancers. Even small cell cancer of the lung, initially chemotherapy sensitive, tends to return after remission, with widespread metastatic spread leading to death of the patient. Thus, better treatment approaches are needed for this illness. The biology of malignant melanomas offers an example of the importance of transcription factors for malignant cell propagation. Malignant melanomas have great propensity to metastasize and are notoriously resistant to conventional cancer treatments such as chemotherapy and γ-irradiation. Development of malignant melanoma in humans progresses through a multistage process, with transition from melanocyte to nevi, to radial growth, and subsequently to the vertical growth, metastatic phenotype of autonomous melanomas, associated with decreased dependence on growth factors, diminished anchorage dependence, reduced contact inhibition and increased radiation and drug resistance.
Much of the molecular understanding of melanoma progression has come from studying the response of cultured melanoma cells to mitogenic stimuli. In culture, melanocyte proliferation and differentiation are positively regulated by agents that increase cAMP (Cox, et al., Nucleic Acids Res. 20:4881-4887 (1992); Halaban, et al., J. Cell Biol. 97:480-488 (1983); Jean, et al., J. Biol. Chem. 273:24884-24890 (1998); Klatt, et al., J. Biol. Chem. 274:15857-15864 (1999); Lehmann, et. al., Proc. Natl. Acad. Sci. U.S.A. 89:9891-9895 (1989); Luca, et al., Melanoma Res. 3:35-41(1993); Richards, et al., J. Biol. Chem. 271:13716-13723 (1996); and Xie, et al., Oncogene 15:2069-2075 (1997)), and several cAMP responsive transcription factors binding to CRE (the consensus motif 5′-TGACGTCA-3′, or cAMP response element) play prominent roles in mediating melanoma growth and metastasis. In MeWo melanoma cells, the transcription factor CREB (for CRE-binding protein) and its associated family member ATF-1 promote tumor growth, metastases and survival through CRE-dependent gene expression. Jean, et al., supra. Expression of the dominant negative KCREB construct in metastatic MeWo melanoma cells decreases their tumorigenicity and metastatic potential in nude mice. Xie, et al., Cancer Res. 57:2295-2303 (1997). The KCREB-transfected cells display a significant decrease in matrix metalloproteinase 2 (MPP2, the 72 kDa collagenase type IV) mRNA and activity, resulting in decreased invasiveness through the basement membrane, an important component of metastatic potential.
The cell surface adhesion molecule MCAM/MUC18, which is involved in metastasis of melanoma is also down-regulated by KCREB transfection. Xie, et al., Cancer Res., supra. In addition, expression of KCREB in MeWo cells renders them susceptible to thapsigargin-induced apoptosis, suggesting that CREB and its associated proteins act as survival factors for human melanoma cells, thereby contributing to the acquisition of the malignant phenotype. Jean, et al., supra.
Melanoma cells aberrantly express the major histocompatibility complex class II (MHC II) antigens normally found only in B-lymphocytes and antigen presenting cells of the monocyte/macrophage cell line. Cox, et al., Nucleic Acids Res. 20:4881-4887 (1992). In B16 melanoma cells this is due to activation of the MHC II DRα promoter by constitutive activation of an ATF/CREB motif. CREB family proteins also bind to the UV-response element (URE, 5′-TGACAACA-3′), and URE binding of the CREB family member ATF-2 confers resistance to irradiation and to the chemotherapeutic drugs cis-platinum, 1-β-D-arabinofuranosylcytosine (araC) or mitomycin C in MeWo melanoma lines. Ronai, et al., Oncogene 16:523-531 (1998). Thus, CREB family transcription factors play important roles in the malignant potential of this tumor type. Thus, targeted molecular disruption of ATF/CREB-mediated transcription might be therapeutically useful for controlling growth and metastases of relatively treatment-resistant malignant melanoma. Jean, supra, and Ronai, supra.
Transcription factors would seem particularly sensitive to this approach. The positively charged DNA binding domain of many transcription factors contains cysteines that can be oxidatively modified by agents such as hydrogen peroxide or nitric oxide (NO*), stimulating repair processes that result in formation of mixed disulfides between glutathione (GSH) and protein thiols. As a consequence of this so-called protein “S-glutathionylation”, the usually positively charged transcription factor DNA binding domain develops an electronegative charge imparted by dual carboxylate end groups of GSH. The change in charge disrupts transcription factor binding to its respective DNA consensus sequence. Sies, Free Rad. Biol. Med. 27:916-921 (1999); Klatt, et al., supra. This mechanism explains how NO* inhibits c-Jun DNA binding by specifically targeted S-glutathionylation of cysteines within the DNA binding region, and a similar mechanism has been suggested for how nitrosative stress functionally inhibits the activity of Fos, ATF/CREB, Myb and Rel/NF-κB family transcription factors. See, Klatt, et al., supra. The transcription factors nuclear factor-κB (NF-κB), activator protein-1 (AP-1) and ATF/CREB all contain cysteines in their DNA binding regions as reactive sites for mixed disulfide formation. Klatt, et al., supra; Pineda-Molina, et al., Biochem. 40:14134-14142 (2001); Marshall, et al., Biochem. 40:1688-1693 (2001); Nikitovic, et al., Biochem. Biophys. Res. Commun. 242:109-112 (1998); Goren, et al., J. Mol. Biol. 313:695-709 (2001); Richards, et al., J. Biol. Chem. 271:13716-13723 (1996).
One therapeutic use for this approach is treatment of malignant melanoma. Melanomas are dependent for growth and metastasis on activation of distinct transcription factors, such as NF-κB (Yang, et al., Cancer Res. 61:4901-4909(2001)), and ATF/CREB transcription factors binding to the cyclic AMP response element CRE (Xie, et al., Oncogene, supra); Jean, et al., supra; Xie, et al., Cancer Res., supra; Cox, et al., Nucleic Acids Res. 20:4881-4887 (1992); Ronai, et al., Oncogene 16:523-531 (1999)). Targeted molecular disruption of ATF/CREB-mediated transcription has been proposed as a strategy to control melanoma growth and metastasis (Jean, et al., supra; Ronai, et al., supra).
The dithiocarbamate disulfide, disulfiram, was once used as a treatment for alcoholism, but has been reported to reverse in vitro resistance of human tumors to chemotherapy drugs by blocking maturation of the P-glycoprotein membrane pump that extrudes chemotherapeutic agents from the cell. Loo, et al., J. Natl. Cancer Inst. 92:898-902 (2000). Disulfiram also enhances efficacy of 5-fluorouracil against human colorectal cancer cell lines (Wang, et al., Cancer Res. 43:954 (abstract) (2002)), inhibits DNA topoisomerases (Yakisch, et al., Biochem. Biphys. Res. Commun. 289:586-590 (2002)), induces apoptosis in cultured melanoma cells (Cen, et al., Mol. Cancer Therapeut. 1:197-204 (2002)), and reduces angiogenesis and inhibits growth of C6 glioma and metastases of Lewis lung carcinoma in mice (Marikovsky, et al., Int. J Cancer 97:34-41 (2002)).
Dithiocarbamates comprise a broad class of compounds possessing a RR′NC(S)SR″ functional groups, having the ability to complex metals (Nobel, et al., J. Biol. Chem. 270:26202-26208 (1995)) and react with sulfhydryl groups (Orrenius, et al., Biochem. Soc. Trans. 24:1032-1038 (1996)) and glutathione (Burkitt, et al., Arch. Biochem. Biophys. 353:73-84 (1998)). After oxidation by copper ions to their corresponding disulfides, dithiocarbamates inhibit critical sulfhydryls in proteins such as cysteine proteases by forming mixed disulfides with thiols (Nobel, et al., Chem. Res. Toxicol. 10:636-643 (1997)).
It is therefore an object of the present invention to provide a method for the inhibition of ATF/CREB growth to treat certain cancers.
Another object of the present invention is to provide a method for treating certain cancers by administering to a patient a therapeutically effective amount of a thiuram disulfide and a heavy metal ion.
Yet another object of the present invention is to provide pharmaceutical compositions for the treatment of cancer.
It is still another object of the present invention to provide a relatively less toxic agent available for use alone or in combination with current drugs in order to better treat cancer patients without risking injury from the therapy itself.